Immunoassay for quantification of an unstable antigen selected from BNP and proBNP

ABSTRACT

The present invention relates to an immunoassay for detection of BNP, proBNP and fragments thereof. Essentially the assay comprises: a) contacting the antigen with a first antibody specific to a fragment corresponding to amino acids 11-22 of BNP, or to a part of this peptide comprising at least three amino acids of said sequence, to obtain a first order immune complex. b) contacting the first order immune complex obtained at step (a) with a second antibody recognizing said first order immune complex, to obtain a second order immune complex, wherein said antibody is unable to recognize free BNP, proBNP or free first antibody; c) Detecting the second order immune complex.

This application is the National Phase of PCT/FI2008/050184 filed onApr. 14, 2008, which claims priority under 35 U.S.C. 119(e) to U.S.Provisional Application No. 60/911,603 filed on Apr. 13, 2007, and under35 U.S.C. 119(a) to patent application Ser. No. 20075251 filed inFinland on Apr. 13, 2007, all of which are hereby expressly incorporatedby reference into the present application.

FIELD OF THE INVENTION

The present invention relates to immunoassays, and provides animmunoassay method for detection of unstable antigens. The method isspecifically suitable for detection of BNP, proBNP and fragments thereof

BACKGROUND OF THE INVENTION

BNP and proBNP are reliable markers of heart failure (HF) widely used inclinical practice. Several types of sandwich immunoassays (conventionalassays) utilizing two mono- or polyclonal antibodies, specific todifferent epitopes of BNP or BNP-fragment of proBNP molecule aredescribed in literature.

BNP molecule is known as an extremely unstable molecule rapidly losingits immunological activity in water solutions. This loss of activity isusually associated with proteolytic degradation of the peptide. Sandwichimmunoassays commonly used for qualitative or quantitative antigenimmunodetection utilize two or more antibodies specific to two or moredifferent epitopes. The longer is the distance between the epitopes, thehigher is the probability that sites of proteolysis would be locatedbetween the epitopes of the antibodies, thus increasing the sensitivityof the assay to proteolytic degradation of the antigen. And vice versa,the closer are the epitopes to each other, the smaller is theprobability of the proteolytic cleavage of the molecule between theepitopes.

Immunoassay methods for very small molecules have been described,including the application of so called anti-metatype antibodies. Suchmethods are disclosed, e.g. for detecting digoxin (Self et al., 1994,Clin. Chem. 40:2035-2041), and angiotensin II (Towbin et al., 1995, J.Immunol. Meth. 181:167-176).

However, it is not an easy task to apply this type of method todifferent analytes, since very specific monoclonal antibodies arerequired in such a method.

DESCRIPTION OF THE INVENTION

Here we are describing an immunoassay for quantification of BNP andproBNP in human blood. We have named the assay as “unequal sandwich”.This assay is applicable to immunodetection of all unstable antigens.

The immunoassay described in the present application utilizes twodifferent monoclonal antibodies. In detection of BNP or proBNP the firstmonoclonal antibody (MAb 24C5) or fragment thereof, is specific to theregion (or a part of this region) comprising amino acid residues 11-22(₁₁FGRKMDRISSSS₂₂) (SEQ ID NO:3, i.e. amino acids 11-22 of SEQ ID NO:1)of BNP (which correspond to amino acid residues 87-98 of proBNP) (FIG.1). The second antibody (namely MAbs Ab-BNP2 and Ab-BNP4), or fragmentthereof, labeled with a signal-producing component, recognizes an immunecomplex of the first antibody, or fragment thereof, with antigen (BNP,proBNP, or a fragment thereof, comprising amino acid residues₁₁FGRKMDRISSSS₂₂ (amino acids 11-22 of SEQ ID NO:1) or a part of thissequence comprising at least three amino acid residues of saidsequence). The second antibody, or fragment thereof, does not recognize(or recognizes with very low affinity-10—fold or less) either freeantigen or its fragments, or free MAb 24C5, or fragment thereof. Thusthe primary immune complex comprising MAb 24C5, or fragment thereof, andBNP (or proBNP, or a fragment thereof) serves as an antigen for thesecond antibody (MAbs Ab-BNP2 and Ab-BNP4), or fragment thereof

Consequently, the general object of the present invention is animmunoassay method for detecting an unstable antigen in a sample,comprising

-   -   (a) contacting an antigen of interest with a first antibody        specific to a first epitope of the antigen molecule, to obtain a        first order immune complex,    -   (b) contacting the first order immune complex obtained at        step (a) with a second antibody, which recognizes said first        order immune complex and is specific to a second epitope formed        by the antigen of interest and the first antibody, to obtain a        second order immune complex, wherein said second antibody is        unable to recognize free antigen or a fragment thereof or free        first antibody, or recognizes them with significantly lower        affinity—10-fold or less—than they recognize the first order        immune complex, and    -   (c) detecting the second order immune complex formation.

A specific object of the invention is an immunoassay method fordetecting an antigen selected from the group consisting of BNP, proBNPand a fragment thereof in a sample, comprising

-   -   (a) contacting the antigen with a first antibody specific to the        fragment ₁₁FGRKMDRISSSS₂₂ (SEQ ID NO:3, i.e. amino acids 11-22        of SEQ ID NO:1) of BNP molecule or to a part of this peptide        comprising at least three amino acid residues of said sequence,        to obtain a first order immune complex,    -   (b) contacting the first order immune complex obtained at        step (a) with a second antibody recognizing said first order        immune complex, to obtain a second order immune complex, wherein        said second antibody is unable to recognize free BNP, proBNP or        a fragment thereof or free first antibody, or recognizes them        with significantly lower affinity—10-fold or less—than it        recognizes the first order immune complex, and    -   (c) detecting the second order immune complex formation.

We have succeeded in producing specific monoclonal antibodies applicablein the method of the invention. These antibodies are specific objects ofthe present invention.

Unequal sandwich described herein demonstrates extraordinaryinsusceptibility to proteolytic degradation of the antigen in comparisonwith the assays utilizing antibodies specific to distantly locatedepitopes.

Also such approach could be useful in the cases where the assay isdeveloped for immunodetection of the antigen which is similar to one ormore other antigens; has numerous different epitopes on its surface, buthas only one (or more, but very limited number) of unique epitopes, thatdistinguishes that particular antigen from all others.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. BNP and pro BNP structures and epitope specificity of MAb 24C5.

MAb 24C5 recognizes fragment of BNP molecule comprising amino acidresidues 11-22 of SEQ ID NO:1 and proBNP fragment consisting of aminoacid residues 87-98 of SEQ ID NO:2(marked by dark).

FIGS. 2A, 2B and 2C. Antibodies Ab-BNP2 and Ab-BNP4 do not recognizeeither BNP or proBNP that are not complexed with MAb 24C5.

Eu-labelled MAbs 24C5, Ab-BNP2, Ab-BNP4 (200 ng/well) were incubated inplates coated with:

-   -   A. BNP 50 ng/well    -   B. proBNP 100 ng/well    -   C. polyclonal anti-BNP antibodies (2 μg/well) preincubated with        BNP (0.5 ng/well)

FIG. 3. Antibodies Ab-BNP2 and Ab-BNP4 can recognize immune complex ofBNP (or Peptide 11-22) with MAb 24C5

Three-step assay protocol:

First step: plates were precoated with capture MAb 24C5

Second step: After washing the plates were incubated with antigen (BNPor Peptide 11-22);

Third step: After washing the plates were incubated with detection (Eu³⁺labeled) antibodies (Ab-BNP2, Ab-BNP4 or 57H3).

After washing enhancement solution was added and the signal wasmeasured.

FIG. 4. Antibodies Ab-BNP2 and Ab-BNP4 can recognize proBNP, which formsimmune complex with MAb 24C5

Three-step assay protocol:

First step: Plates were precoated with capture MAb 24C5

Second step: After washing the plates were incubated with proBNP (5ng/ml)

Third step: After washing the plates were incubated with detectionantibodies (Ab-BNP2, Ab-BNP4 or 57H3).

After washing enhancement solution was added and the signal wasmeasured.

FIG. 5. Stability of BNP in normal human plasma.

Synthetic BNP was spiked into pooled normal human plasma (2 ng/ml),incubated at +4° C. for different periods of time. Immunologicalactivity was tested in three different assays—one conventional and twounequal sandwiches.

FIG. 6. BNP/proBNP measurements in blood of patients with HF and healthydonors. Plasma samples of 6 patients with heart failure (HF 1-HF 6) andplasma samples of healthy donors (NP1-NP4) were tested in three assays.Synthetic BNP (Bachem) was used as a calibrator in all assays.

FIGS. 7A, 7B and 7C. Calibration curves for two unequal sandwiches(24C5-Ab-BNP2, 24C5-Ab-BNP4) and one conventional assay (50E1-24C5-Eu).Antigen: synthetic BNP (Bachem).

EXPERIMENTAL

Remarks: Antibodies labeled with stable Eu-chelate were used in allexperiments as detection antibodies. The monoclonal antibodies 24C5,Ab-BNP2, Ab-BNP4, 57H3 and 50E1 used in the experiments are availablefrom Hytest Ltd, Turku, Finland.

EXAMPLE 1 Antibodies Ab-BNP2 and Ab-BNP4 Do Not Recognize Either BNP orproBNP That Are Not Complexed with MAb 24C5 (FIG. 2)

In the experiment presented in the FIG. 2A and FIG. 2B antigens (BNP andproBNP, respectively) were used for plate coating and Eu-labeledantibodies were tested with the antigen in direct immunoassay. Antibody24C5 recognizes both forms of the antigen, whereas MAbs Ab-BNP2 andAb-BNP4 give no response (signal comparable with background) with any ofthe two antigens.

In the experiment presented in FIG. 2C the plates were coated withpolyclonal antibodies specific to different epitopes on BNP molecule. Onthe second step the plates were incubated with BNP and then withEu-labeled antibodies. Such approach helps to obtain variableorientation of the antigen against plate surface, insuring thatorientation of the molecule on the plate surface does not have influenceon the experimental results. In this experiment the same results asdescribed above were obtained: MAbs Ab-BNP2 and Ab-BNP4 were not able torecognize the antigen, which is not complexed with MAb 24C5.

EXAMPLE 2 Antibodies Ab-BNP2 and Ab-BNP4 Can Recognize BNP and Peptide11-22, that are Forming Immune Complex with MAb 24C5 (FIG. 3)

MAb 24C5 is specific to the fragment 11-22 of BNP molecule or to thecorresponding region 87-98 of proBNP. To demonstrate that immune complex24C5-BNP and 24C5-peptide 11-22 could be recognized by MAbs Ab-BNP2 andAb-BNP4 we used MAb 24C5 for plate coating, then incubated the plateswith BNP or synthetic peptide corresponding to amino acids 11-22 of BNPsequence (Peptide 11-22). After the immune complex between MAb 24C5 andantigens was formed, the plates were incubated with Eu-labeledantibodies Ab-BNP2, Ab-BNP4 and 57H3, specific to the region 26-32 ofthe BNP molecule.

Unequal sandwich recognizes BNP and the peptide almost with the sameefficiency. Assay utilizing antibodies 24C5 (coating)—57H3-Eu does notrecognize Peptide 11-22 (signal comparable with the background).

EXAMPLE 3 Antibodies Ab-BNP2 and Ab-BNP4 Can Recognize proBNP, WhichForms Immune Complex with MAb 24C5 (FIG. 4)

Unequal sandwich recognizes proBNP with the same efficiency as aconventional assay. We used MAb 24C5 for plate coating and thenincubated plates firstly with recombinant proBNP (5 ng/ml) and secondlywith Eu-labeled antibodies Ab-BNP2, Ab-BNP4 and 57H3 specific to theregion 26-32 of BNP molecule. The signals obtained in the unequalsandwich and conventional immunoassays are comparable. We concluded thatnew assays could be used for quantitative immunodetection of proBNP.

EXAMPLE 4 Apparent Stability of the Antigen (FIG. 5)

Synthetic BNP (Bachem) was spiked into pooled normal human plasma (2ng/ml), incubated at +4° C. for different periods of time and theimmunological activity was tested in three different assays - oneconventional and two unequal sandwiches.

Apparent stability of the antigen, being determined in unequalsandwiches, described here is significantly higher in comparison withthe stability determined by the conventional BNP assays utilizing twoMAbs specific to different parts of BNP molecule. As an example ofconventional assay we used assay, utilizing MAb 50E1 specific to theregion 26-32 of BNP molecule and MAb 24C5 specific to the region 11-22of BNP molecule. About 70% of immunological activity was observed after24 hours of incubation at +4° C. (69.8% and 68% for assays utilizingAb-BNP2 and Ab-BNP4, respectively) in the case the unequal sandwich wasused to determine the immunoreactivity, and only 28% in the case ofconventional assay. Six days after the beginning of incubation noimmunoreactivity was observed in case of conventional assays, whereasabout ¼ of initial immunoreactivity was observed in the case of unequalsandwiches.

EXAMPLE 5 BNP/proBNP Measurements in Blood of Heart Failure Patients (HFPatients) and Blood of Healthy Donors (FIG. 6)

Unequal sandwich, as well as conventional BNP assays are able to detectin human blood both forms of the antigen displaying “BNPimmunoreactivity”—i.e. BNP and proBNP. Blood samples from several HFpatients and healthy donors were tested in three assays—oneconventional, utilizing capture MAb 50E1, specific to the fragment 26-32of BNP molecule and detection MAb 24C5-Eu and two unequal sandwiches.All assays were calibrated using synthetic BNP. As it follows from FIG.6, the results of testing in three assays are very similar. In somesamples results of testing in conventional assay are lower than inunequal sandwiches. This observation can be explained by the fact thatin such samples BNP is partially degraded, but because of the fact thatantigen displays better apparent stability in unequal sandwiches theantigen values determined by these assays are higher than in aconventional assay.

EXAMPLE 6 Calibration Curves (FIG. 7)

Calibration curves for two unequal sandwiches and one conventional assaywith synthetic BNP used as an antigen are presented in FIGS. 7 (A, B andC). Both of the unequal sandwiches demonstrate high sensitivity,comparable with the sensitivity of the conventional assay and could beused for precise detection of BNP and proBNP immunoreactivity in humanblood.

The invention claimed is:
 1. An isolated monoclonal antibody, or bindingfragment thereof, specific to an epitope located on a first order immunecomplex, the first order immune complex consisting of a first antibody,or binding fragment thereof, bound to BNP, to proBNP, or to a fragmentof BNP or proBNP, the first antibody being specific for a regionconsisting of the amino acid sequence ₁₁FGRKMDRISSSS₂₂ (SEQ ID NO:3) ofthe BNP or proBNP protein, or to a portion of the region, the epitopeforming when the first antibody, or binding fragment thereof, and theBNP or proBNP protein, or a fragment of the BNP or proBNP protein, arein the first order immune complex, wherein the isolated monoclonalantibody, or binding fragment thereof, does not recognize free BNP, freeproBNP, a fragment of BNP or proBNP, or free first antibody, or abinding fragment thereof, or which recognizes them with 10-fold or lessaffinity than it recognizes the first order immune complex, wherein theisolated monoclonal antibody or binding fragment thereof is Ab -BNP2,Ab-BNP4, or a binding fragment thereof.